Volumetric analysis of epithelial morphogenesis with high spatiotemporal resolution

项目摘要 上皮片如何通过改变邻近的细胞来重塑自己以采用新的组织构象 关系和细胞形状动力学一直是发育和疾病中的关键问题。有趣的是， 在模型系统中进行的许多开创性的研究在很大程度上局限于二维分析， 常常受到挑战，以成像发生在位于组织更深处的基底区域中的细胞行为。 在果蝇原肠胚的组织伸长过程中发生的插入运动是一个经典的 系统了解上皮重塑，并已基本告知发育 描述细胞如何在粘附上皮中改变位置的范例。几乎所有的研究， 该系统仅限于早期苍蝇胚胎顶端事件的2D分析，迄今为止还没有研究 系统地分析了驱动上皮重塑和组织延伸的完整3D行为， 果蝇胚胎上皮。因此，该领域最大的遗留问题之一是， 上皮细胞的体积性质影响力的传播和细胞表面的重塑，沿着整个 顶基轴关于力从哪里来以及力有多远和多快的基本问题 在不同的顶-基底层之间传播的问题仍然没有答案。根据初步分析，我们 已经成功地完成了第一个完整的4D分割的嵌入果蝇上皮细胞 使用Lattice Light Sheet Microscopy（LLSM）。我们发现，嵌入可以在任何 我们已经沿着顶-基轴沿着测量的位置。这是惊人的，因为以前的研究主要是 涉及心尖力的产生，一项研究表明，收缩力也可以起源于 从上皮的基底表面。在拟议的项目中，我们正在开发工具来执行 首次对果蝇早期胚胎中的细胞嵌入进行全面的定量3D分析。然后我们将 确定驱动3D力产生的分子机制，以及不同的机械机制是否 存在于顶基轴上。初步数据表明，高度新颖的动态肌球蛋白II和F-肌动蛋白 在侧部和基部区域表现出快速轴向繁殖的种群。这些的3D分布 人群正在绘制，相关的肌动蛋白成核和肌球蛋白调控网络将被 测定这些结果将提供第一次全面了解皮质和收缩 决定原肠胚上皮的机械环境的网络。我们还将使用3D数据 设置在野生型和功能受损的背景，以检查上皮部队如何传播沿着 使用拓扑映射度量的顶部-底部和平面尺寸。我们将决定距离多远， 速度，收缩力在完整的，发育中的上皮中传播。这些结果将提供基本的 回答了粘性细胞质和弹性细胞皮层如何响应力驱动的位移， 这些位移如何在单个细胞内和组织间传播，以驱动新的组织拓扑结构。

Project Summary How epithelial sheets remodel themselves to adopt new tissue conformations through changes in neighbor relationships and cell shape dynamics has been a key question in development and disease. Interestingly, many of the pioneering studies performed in model systems have largely been confined to 2D analysis, and have often been challenged to image cell behaviors that occur in basal regions that lie deeper into the tissue. The intercalation movements that occur during tissue elongation in the Drosophila gastrula have been a classic system for understanding epithelial remodeling, and have been fundamental to informing the developmental paradigms that describe how cells can change position in an adherent epithelium. Nearly all of the studies in this system have been confined to 2D analysis of apical events in the early fly embryo, and no studies to date have systematically analyzed the full 3D behaviors that drive epithelial remodeling and tissue extension in the Drosophila embryonic epithelium. Thus, one of the biggest remaining questions in the field is how the volumetric nature of epithelial cells affects force propagation and remodeling of the cell surface along the entire apical-basal axis. Fundamental questions on where forces originate from as well as how far and fast forces propagate across different apical-basal layers have remained unanswered. In our preliminary analysis, we have been successful in completing the first full 4D segmentation of the intercalating Drosophila epithelium through the use of Lattice Light Sheet Microscopy (LLSM). We find that intercalation can be initiated at any position we have surveyed along the apical-basal axis. This is striking as previous studies have largely implicated apical force generation, and a single study has suggested that contractile forces can also originate from the basal surface of the epithelium. In the proposed project, we are developing the tools to perform the first comprehensive, quantitative 3D analysis of cell intercalation in the early Drosophila embryo. We will then determine the molecular mechanisms driving 3D force generation, and whether different mechanical regimes exist across the apical-basal axis. Preliminary data suggests highly novel dynamic Myosin II and F-actin populations that show rapid axial propagation in lateral and basal regions. The 3D distributions of these populations are being mapped and the relevant actin nucleating and Myosin regulatory networks will be determined. These results will provide the first comprehensive understanding of the cortical and contractile networks that determine the mechanical environment of a gastrulating epithelium. We will also use 3D data sets in wild-type and functionally compromised backgrounds to examine how epithelial forces propagate along apical-basal and planar dimensions using topological mapping metrics. We will determine how far, and at what velocities, contractile forces spread in an intact, developing epithelium. These results will give fundamental answers into how the viscous cytoplasm and elastic cell cortex respond to force-driven displacements, and how these displacements spread within individual cells and across tissues to drive new tissue topologies.